DNA damage and apoptosis in the mussel Mytilus galloprovincialis

The effects of known genotoxic substances (4-nitroquinoline-N-oxide, benzo[a]pyrene, teniposide, etoposide, cycloheximide, tributyltin) on human cells (FLC, HL-60) and on mussels were investigated. The correlations between formation of DNA strand breaks and DNA fragmentation characteristic for the process of apoptosis were estimated. Strand breaks induced by 4-nitroquinoline-N-oxide and benzo[a]pyrene did not correlate with DNA fragmentation detected in the process of apoptosis. Induction of internucleosomal DNA fragmentation in HL-60 cells was initiated by teniposide, etoposide and tributyltin, while in the gills of mussels this was detected only with tributyltin. Levels of DNA strand breaks in natural mussel populations, living at locations under the influence of urban and industrial wastes, do not mirror the apoptotic processes.     

Effects of 17beta-estradiol exposure in the mussel Mytilus galloprovincialis

Mussels Mytilus galloprovincialis were exposed to different concentrations of estradiol (20, 200, and 2000 ng/l) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels dealt with exogenous estrogenic compounds. Sex hormone levels were determined in whole tissue. Free-estradiol was only significantly elevated at the highest exposure dose (up to 10-fold). Most of the estradiol was in the tissues as fatty acid esters (> 78%), which sharply increased in a dose-dependent manner (from 4 ng/g in controls to 258 ng/g at the high exposure group). In contrast, neither free nor esterified testosterone levels showed significant differences between control and exposure groups. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal status, and the important role that fatty acid esterification may play within those mechanisms. Synthesis and conjugation rates of estradiol were further investigated by measuring the activity of P450 aromatase, and palmitoyl-CoA:estradiol acyltransferase, in digestive gland microsomal fractions. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.     

Colloidal complexed silver and silver nanoparticles in extrapallial fluid of Mytilus edulis

Metal transport in mollusk extrapallial fluid (EPF) that acts as a "bridge" between soft tissues and shell has surprisingly received little attention until now. Using ultrafiltration and radiotracer techniques we determined silver concentrations and speciation in the EPF of the blue mussel Mytilus edulis after short-term uptake and depuration laboratory experiments. Radiolabelled silver ((11?m)Ag) was used in dissolved or nanoparticulate phases (AgNPs < 40 nm), with a similar low Ag concentration (total radioactive and cold Ag ~0.7 μg/L) in a way that mussels could uptake radiotracers only from seawater. Our results indicated that silver nanoparticles were transported to the EPF of blue mussels at a level similar to the Ag ionic form. Bulk activity of radiolabelled silver in the EPF represented only up to 7% of the bulk activity measured in the whole mussels. The EPF extracted from mussels exposed to both treatments exhibited an Ag colloidal complexed form based on EPF ultrafiltration through a 3 kDa filter. This original study brings new insights to internal circulation of nanoparticles in living organisms and contributes to the international effort in studying the potential impacts of engineered nanomaterials on marine bivalves which play an essential role in coastal ecosystems, and are important contributors to human food supply from the sea.     

Embryotoxicity of TiO2 nanoparticles to Mytilus galloprovincialis (Lmk)

Few data exist on the ecotoxicological effects of nanosized titanium dioxide (nTiO₂) towards marine species with specific reference to bivalve molluscs and their relative life stages. Mytilus galloprovincialis Lamarck was selected to assess the potential adverse effects of nTiO₂ (0–64 mg/L) on its early larval development stages (pre-D shell stage, malformed D-shell stage and normal D-shell stage larvae) considering two exposure scenarios characterised by total darkness (ASTM protocol) and natural photoperiod (light/dark). This approach was considered to check the presence of potential effects associated to the photocatalytic properties of nTiO₂. Parallel experiments were carried on with the bulk reference TiCl₄. The toxicity of nTiO₂ showed to be mainly related to its “nano” condition and to be influenced by the exposure to light that supported the increase in the number of pre-D shell stage (retarded) larvae compared to the malformed ones especially at the maximum effect concentrations (4 and 8 mg nTiO₂/L). The non-linear regression toxicity data analysis showed the presence of two EC50 values per exposure scenario: a) EC(50)₁ = 1.23 mg/L (0.00–4.15 mg/L) and EC(50)₂ = 38.56 mg/L (35.64–41.47 mg/L) for the dark exposure conditions; b) EC(50)₁ = 1.65 mg/L (0.00–4.74 mg/L) and EC(50)₂ = 16.39 mg/L (13.31–19.48 mg/L) for the light/dark exposure conditions. The potential implication of agglomeration and sedimentation phenomena on ecotoxicological data was discussed.     

Effect of pollution on genetic diversity in the bay mussel Mytilus galloprovincialis and the acorn barnacle Balanus glandula.

To test if environmental contamination acts as a selection force affecting genetic diversity at the population level, two intertidal invertebrate species, Mytilus galloprovincialis and Balanus glandula, were collected from seven different bay sites in southern California. Collections were made at three relatively pristine 'clean' sites and four 'impacted' sites exposed to heavy industrial or boating activity, and which had previously been identified as having measurable levels of pollution. Genetic diversity at each site was assessed by comparing fragment polymorphisms generated from genomic DNA by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). All populations retained a large amount of genetic diversity and were genetically similar to each other. However, several different measures of diversity indicated that, for most primers, the populations of both species from impacted sites had lower genetic diversity compared to those populations from clean sites. Individuals at impacted sites were more likely to share the same haplotypes than were those from clean sites. Few bands seen in the clean sites were absent from the impacted sites or vice versa, but a number of bands in the clean site populations were significantly less common in the impacted populations, while a few bands uncommon in clean site populations were more common at impacted sites. Together, these results suggest that pollution at the impacted sites may reduce genetic diversity among the resident invertebrate populations.     

基于线粒体COI序列分析对紫贻贝群体遗传多样性的研究分析

本文采用线粒体COI基因序列分析对我国沿海6个紫贻贝群体124个个体的遗传多样性和群体结构进行了初步研究.在紫贻贝6个群体中共发现13个单倍型和33个核苷酸多态位点.青岛群体单倍型数目是最多的,温州群体、丹东群体和宁德群体次之.6个紫贻贝群体的基因多样性为0.569～0.821,核苷酸多样性为0.015 5～0.051...     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6beta-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r2 = 0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

Colonisation of South African kelp-bed canopies by the alien mussel Mytilus galloprovincialis: extent and implications of a novel bioinvasion

The Mediterranean mussel Mytilus galloprovincialis is the most significant invasive alien marine species in South Africa and, although not normally found subtidally, has recently been observed colonising heads and stipes of the kelp species Ecklonia maxima in False Bay. We quantified this invasion and explored its ecological implications. Transects laid across kelp beds revealed that 10.34% of kelp individuals surveyed bore canopy mussels, with kelp heads (10.07%) being far more commonly infected than stipes (0.27%). Twenty kelp individuals with infected heads and 20 with infected stipes were separately collected for more-detailed examination. Wet mass of mussels on these kelp heads ranged from 2.5 to 2 462 g (median 86.4 g, interquartile range 14.8–353.8 g) and that of mussels on the stipes from 7.6 to 3 492 g (median 595.5 g, interquartile range 194.0–955.0 g). Mussel clumps consisted mostly of individuals <40 mm in length. Mussel clumps supported a rich biota of 80 invertebrate and 13 algal species. Larger clumps supported more epibiotic species, and those on stipes more species than those of comparable mass on kelp heads. The mussels and their associated epibiotic species negatively affected kelp buoyancy, but rarely enough to overcome natural buoyancy. Some kelp individuals that had been toppled by the weight of mussels and their epibiotic species, however, were encountered in situ. Implications of this invasion include large increases in animal biomass and species richness in the kelp canopy, plus reductions in kelp buoyancy and increased hydrodynamic drag on infected kelps, increasing their probability of being uprooted. Uprooted kelp individuals can raft long distances, potentially transporting both native and alien species to distant sites.     

Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy)

A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments.     

Variability of the hemocyte parameters of cultivated mussel Mytilus galloprovincialis (Lmk 1819) in Sabaudia (Latina,Italy) coastal lagoon

The Sabaudia's lake consists of a protected coastal lagoon, located in the central Italy, historically characterized by recurrent mortality events of marine fauna during warmer months. A field study was monthly conducted on mussels Mytilus galloprovincialis cultivated inside the lagoon, measuring hemocyte parameters as total circulating count (THC), viability (HV), spreading and oxidative response to in vitro phagocytosis stimulation. A depression of the immune response was observed during the spring season, as indicated by higher values of hemocyte circularity and lower luminescence levels related to respiratory burst, also associated to modulation of THC and HV. The water temperature and the oxygen concentration appeared as the major environmental factors having influence on the phagocytosis activity. Therefore, the hemocyte variations have been intended as early danger signal to evaluate the immunodepression induced by the environmental stressors which could reveal in advance the development of critical situations for mussel survival.     

Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.     

Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis.

Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux. This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay. The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages. The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater. Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil. Larval growth was also significantly impaired in the presence of verapamil. Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive. This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.     

舟山海域外来物种地中海贻贝的自然分布现状及生态影响

以筛选的2个可鉴别的地中海贻贝（Mytilus galloprovincialis）、厚壳贻贝（M.coruscus）及其杂交后代的PCR标记（扩增核基因Glu-5’特异片段PCR标记Me14/Me17和延伸因子1α第一个内含子区段PCR标记引物EFbisF/EFbisR）,对舟山海域3个贻贝主产区（马鞍列岛海域、浪岗山列岛海域、中街山列岛海域）采集的贻贝样本的种类进行鉴别.研究结果显示：100个马鞍列岛海域贻贝个体和30个中街山列岛海域贻贝个体均为厚壳贻贝;而54个浪岗山列岛海域贻贝样本中,16个个体（29.6%）为厚壳贻贝,38个个体（70.4%）为地中海贻贝;3个群体中均未检测到2种贻贝的杂交个体.因此,推测舟山海域地中海贻贝自然分布仅局限于局部海区,但在适宜的海区地中海贻贝可取代厚壳贻贝成为贻贝床的优势物种,舟山海域贻贝床的动态变化值得进一步关注;贻贝样本中没有检测到地中海贻贝与厚壳贻贝的杂交个体,表明调查海域可能没有杂交贻贝分布或数量极少,地中海贻贝通过杂交、基因渐渗污染厚壳贻贝基因组的风险较小.     

Phase I and II biotransformation enzymes and polycyclic aromatic hydrocarbons in the Mediterranean mussel (Mytilus galloprovincialis,Lamarck, 1819) collected in front of an oil refinery

The aim of the present study was to investigate the responses of phase I and II biotransformation enzymes and levels of PAHs in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected from three sites at different distance from an oil refinery. Phase I enzyme activities as NAD(P)H-cyt c red, NADH ferry red, B(a)PMO and phase II as UDPGT, GST were measured in digestive gland while 16 PAHs (US-EPA) in whole soft tissue. An added value to the data obtained in the present study rely on the RDA analysis which showed close correlations between PAHs levels and phase I enzyme activities in mussels collected in front of the refinery. And again a significant spatial correlation between B(a)P levels and NADPH-cyt c red activities was observed using linear models. No differences among sites for B(a)PMO and phase II GST activities were observed, while the application of UDPGT as biomarkers requires further investigation.     

Gold nanoparticles and oxidative stress in Mytilus edulis

Little is known about potential environmental impact of nanoparticles. Gold nanoparticles can cause unexpected biological responses. Here, Mytilus edulis were exposed (24h) to gold-citrate nanoparticles (GNP), menadione and both compounds simultaneously (GNP/menadione). Protein ubiquitination and carbonylation were determined in gill, mantle and digestive gland, along with traditional oxidative stress biomarkers; catalase activity and neutral red retention time assay (haemolymph). 2DE gels were performed on gill proteins (menadione; GNP/menadione). Our results reveal that GNP may induce oxidative stress.     

Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium

Although the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5 +/- 0.5 cm and large: 5.2 +/- 0.7 cm) were exposed to Cd (100 microg l(-1)) at different water temperatures (5, 18 and 25 degrees C) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 degrees C) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 degrees C, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 degrees C there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.     

基于紫贻贝Mytilus galloprovincialis表达序列的核受体家族分析

通过对紫贻贝(Mytilus galloprovincialis)公共数据库表达序列的整合分析,共获得29条核受体序列。通过DNA结合结构域(DBD)和配体结合结构域(LBD)的多序列比对,并采用贝叶斯推断法进行系统进化分析,推测其中19个核受体属于NR1亚家族,5个属于NR2亚家族,2个属于NR3亚家族,1个属于NR5亚家族。结果表明紫贻贝中存在HR96、E75、ROR、HNF4、RXR、TLL、SVP、ESR、ERR同源的核受体,并且具有新型的2DBD核受体。